Fig. 7: Resistant HIV-1 mutants arise in T cells expressing the TAR2 or MA sgRNA but not in cells expressing the TAR1 sgRNA.

(A) Representative growth curve measuring the level of intracellular NLuc expression seen in SupT1 cells stably expressing the Sp Cas9 protein and a control (Ctrl) or HIV-1 specific sgRNA (MA, TAR1 or TAR2). Cells were infected with a stock of the NL-NLuc-HXB indicator virus on day 0 and then passed into fresh media every four days, at which point an aliquot of 3 × 10⁵ cells was harvested and used to measure intracellular NLuc expression, as indicated. Arrows indicate the times when supernatant media were sampled to detect released, infectious HIV-1. (B) Media sampled from the SupT1 cells in panel A at day 12 were used to infect fresh Cas9-expressing SupT1 cells expressing the cognate sgRNA, as indicated. Cells were then harvested 4 days later and intracellular NLuc levels determined. (C) Same as panel B except using supernatant media harvested at day 16.